Sundram Vasudha, Nanda Jagpreet S, Rajagopal Kammara, Dhar Jayeeta, Chaudhary Anita, Sahni Girish
Institute of Microbial Technology, Chandigarh 160036, India.
J Biol Chem. 2003 Aug 15;278(33):30569-77. doi: 10.1074/jbc.M303799200. Epub 2003 May 27.
To explore the interdomain co-operativity during human plasminogen (HPG) activation by streptokinase (SK), we expressed the cDNAs corresponding to each SK domain individually (alpha, beta, and gamma), and also their two-domain combinations, viz. alphabeta and betagamma in Escherichia coli. After purification, alpha and beta showed activator activities of approximately 0.4 and 0.05%, respectively, as compared with that of native SK, measured in the presence of human plasmin, but the bi-domain constructs alphabeta and betagamma showed much higher co-factor activities (3.5 and 0.7% of native SK, respectively). Resonant Mirror-based binding studies showed that the single-domain constructs had significantly lower affinities for "partner" HPG, whereas the affinities of the two-domain constructs were remarkably native-like with regards to both binary-mode as well as ternary mode ("substrate") binding with HPG, suggesting that the vast difference in co-factor activity between the two- and three-domain structures did not arise merely from affinity differences between activator species and HPG. Remarkably, when the co-factor activities of the various constructs were measured with microplasminogen, the nearly 50-fold difference in the co-factor activity between the two- and three-domain SK constructs observed with full-length HPG as substrate was found to be dramatically attenuated, with all three types of constructs now exhibiting a low activity of approximately 1-2% compared to that of SK.HPN and HPG. Thus, the docking of substrate through the catalytic domain at the active site of SK-plasmin(ogen) is capable of engendering, at best, only a minimal level of co-factor activity in SK.HPN. Therefore, apart from conferring additional substrate affinity through kringle-mediated interactions, reported earlier (Dhar et al., 2002; J. Biol. Chem. 277, 13257), selective interactions between all three domains of SK and the kringle domains of substrate vastly accelerate the plasminogen activation reaction to near native levels.
为了探究链激酶（SK）激活人纤溶酶原（HPG）过程中的结构域间协同作用，我们分别表达了与SK每个结构域（α、β和γ）对应的cDNA，以及它们的两个结构域组合，即αβ和βγ，在大肠杆菌中进行表达。纯化后，与天然SK相比，α和β在人纤溶酶存在下测量时，分别显示出约0.4%和0.05%的激活剂活性，但双结构域构建体αβ和βγ显示出更高的辅因子活性（分别为天然SK的3.5%和0.7%）。基于共振镜的结合研究表明，单结构域构建体对“伙伴”HPG的亲和力显著较低，而双结构域构建体在与HPG的二元模式以及三元模式（“底物”）结合方面，其亲和力与天然情况非常相似，这表明两结构域和三结构域之间辅因子活性的巨大差异不仅仅源于激活剂物种与HPG之间的亲和力差异。值得注意的是，当用微纤溶酶原测量各种构建体的辅因子活性时，以全长HPG为底物时观察到的两结构域和三结构域SK构建体之间近50倍的辅因子活性差异被显著减弱，与SK、HPN和HPG相比，所有三种类型的构建体现在都表现出约1 - 2%的低活性。因此，底物通过催化结构域对接至SK - 纤溶酶（原）的活性位点，充其量只能在SK.HPN中产生最低水平的辅因子活性。因此，除了通过先前报道的（Dhar等人，2002；《生物化学杂志》277，13257）kringle介导的相互作用赋予额外的底物亲和力外，SK的所有三个结构域与底物kringle结构域之间的选择性相互作用极大地加速了纤溶酶原激活反应至接近天然水平。
